Ribosomal DNA internal transcribed spacer (ITS2) sequences differentiate Anopheles funestus and An. rivulorum, and uncover a cryptic taxon

Differentiation among the closely related Afrotropical species comprising t he Funestus Group is difficult by traditional taxonomic measures. Anopheles rivulorum is the second most abundant and widespread species in the Funest us Group, and is occasionally collected indoors along with the dominant mem ber and major malaria vector, An. funestus. The prospect of misidentificati on of An. rivulorum as An. funestus prompted the development of a rapid, po lymerase chain reaction (PCR)-based method for identifying these two specie s. The ribosomal internal transcribed spacer 2 (ITS2) was amplified from th irty-five specimens of An. rivulorum collected from the extremes of its ran ge: Eastern Africa (Kenya), Southern Africa (South Africa) and Western Afri ca (Burkina Faso). The ITS2 region of An. rivulorum (approximate to 380 bp) is sufficiently different in size from the ITS2 of An. funestus (approxima te to 700 bp) that these species can be distinguished by agarose gel electr ophoresis of PCR products without further manipulation. Comparison of the A n. rivulorum and An. funestus ITS2 nucleotide sequences revealed such exten sive divergence that meaningful alignment was impossible, except for a 25 b p island near the 5' end. Intraspecific sequence comparisons revealed no va riation among An. rivulorum individuals collected from the same country. Ho wever, sequence divergence was 2% between specimens from South Africa and K enya, and nearly tenfold higher (approximate to 19%) between specimens from Burkina Faso and either South Africa or Kenya, an unprecedented level of i ntraspecific ITS2 divergence in Anopheles. Taken together, these data sugge st that the Burkina Faso sample is not An. rivulorum, but rather a cryptic taxon within the Funestus Group.